http://jla.sagepub.com/Automation

Journal of the Association for Laboratory

http://jla.sagepub.com/content/15/2/107The online version of this article can be found at:

 DOI: 10.1016/j.jala.2009.11.001

2010 15: 107Journal of Laboratory AutomationShifeng Li, Edgar Goluch, Chang Liu, Sandra Szegedi, Kashan Shaikh, Faysal Ahmed, Alan Hu and Shenshen Zhao

Gold Nanoparticle-Based Biodetection for Chip-Based Portable Diagnosis Systems  

Published by:

http://www.sagepublications.com

On behalf of: 

  Society for Laboratory Automation and Screening

can be found at:Journal of the Association for Laboratory Automation Additional services and information for    

  http://jla.sagepub.com/cgi/alertsEmail Alerts:

 

http://jla.sagepub.com/subscriptionsSubscriptions:  

http://www.sagepub.com/journalsReprints.navReprints:  

http://www.sagepub.com/journalsPermissions.navPermissions:  

What is This? 

- Apr 1, 2010Version of Record >>

by guest on October 11, 2013jla.sagepub.comDownloaded from by guest on October 11, 2013jla.sagepub.comDownloaded from by guest on October 11, 2013jla.sagepub.comDownloaded from by guest on October 11, 2013jla.sagepub.comDownloaded from by guest on October 11, 2013jla.sagepub.comDownloaded from by guest on October 11, 2013jla.sagepub.comDownloaded from by guest on October 11, 2013jla.sagepub.comDownloaded from by guest on October 11, 2013jla.sagepub.comDownloaded from

Keywords:

gold nanoparticles,

lab-on-chip,

biobarcode

Original Report

Gold Nanoparticle-BasedBiodetection for Chip-BasedPortable Diagnosis Systems

*CoMeEngEvacha

153

Cop

doi

Shifeng Li, Edgar Goluch, Chang Liu,* Sandra Szegedi, Kashan Shaikh, Faysal Ahmed,Alan Hu, and Shenshen Zhao

MedX Lab, McCormick School of Engineering, Northwestern University, Evanston, IL

We review the past development of highly sensitive

and selective gold nanoparticle (AuNP)-based

assays of protein and DNA biomarkers for chip-based

detection systems. The microfluidic systems, various

assays, and preliminary laboratory results are shown.

AuNP-based biodetection assays provide low detection

threshold, offering promises for multiplexed diagnostics of

many forms of disease markers. ( JALA 2010;15:107–13)

INTRODUCTION

Over the past several years, a number of excitingadvances have been made in the field of nanobio-diagnostics, resulting in assays that rival or surpassthe selectivity and sensitivity of conventional detec-tion methods.1,2 These advances have the potentialto dramatically change the way medical diagnosisand treatments are performed in the future.Through collaboration with Mirkin research groupat the Northwestern University,1,3,4 we have beendeveloping chip-based assays based on biobarcodeassay (BCA) and, more broadly, gold nanoparticle(AuNP)-based assays. The BCA uses AuNPsfunctionalized with oligonucleotides (so-called bio-barcodes, which serve as surrogate targets and

rrespondence: Chang Liu, Ph.D., Professor, Department ofchanical Engineering, MedX Lab, McCormick School ofineering, Northwestern University, 2145 Sheridan Road,nston, IL 60208; Phone: þ1.847.4670701; E-mail:ngliu@northwestern.edu

5-5535/$36.00

yright �c 2010 by The Association for Laboratory Automation

:10.1016/j.jala.2009.11.001

amplifying agents) and a target-recognition element,which may be an antibody for protein detection ora unique oligonucleotide sequence for nucleic aciddetection.1 The BCA also uses functionalized mag-netic microparticles (MMPs), which are adornedwith antibodies that bind to the target. In the pres-ence of targets (protein or oligonucleotide mole-cules) in solution, the MMPs form a sandwichcomplex with the targets and AuNPs, which can belocalized and collected under an applied magneticfield. The barcode oligonucleotide molecules arethen chemically released, identified, and quantified.

The BCA is highly sensitive. In the benchtopformat, the BCA has been shown to achievelow-attomolar sensitivity for protein analytes, upto five orders of magnitude lower than analogousenzyme-linked immunosorbent assay (ELISA) tech-nology, the benchmark method for protein detec-tion. The BCA also has exhibited high-zeptomolarsensitivity for nucleic acid targets, rivaling thePCR-amplification method, which requires thermalcycling. The BCA is also highly specific and capableof extensive multiplexing. Therefore, the BCA offersseveral unique diagnostic opportunities, includingearly disease detection, monitoring of disease recur-rence,5 and the possibility of simultaneous multi-plexed analysis of a panel of disease markers.

Microfluidic systems should and would eventuallyreplace the benchtop assay to achieve portability,automation, and simplicity of use. Our group hasmodified the benchtop BCA to adapt to the micro-fluidic and portable systems requirements. Theseadaptations are very important and often resultsin significant deviation from the benchtop predeces-sors. We have first developed a chip-based BCA.6

Later, this method has been modified to become

JALA April 2010 107

Original Report

the surface-immobilized BCA (SI-BCA),7 which is simplerthan the original BCA and actually work only in the micro-fluid format. Meanwhile, various other detection schemes,such as electronic-gap detection8 and mechanical resonancedetection, have been developed at the proof-of-concept stage.

In terms of microfluidics, our past efforts include manycomponents, including magnetic membrane valves,9,10 mag-netic stir bar mixers,11 flow and channel filling sensors,12

and integrated valves with soft O-ring seals.13 We have alsodeveloped a multilayered modular microfluid architecture14

for low-cost rapid prototyping of custom microfluid systems.

ASSAY DEVELOPMENT

Chip-Based SI-BCA

The BCA was originally developed in a benchtop format.The established benchtop BCA protocol cannot be directlytransferred to a chip-based format because of unique scaling,materials, and microengineering issues. We have successfullydemonstrated a modified BCA that takes advantage of

Figure 1. Schematic diagram of the SI-BCA protocol. (A) The wallsflowed through the capture region. (C) The target molecules attach totagged with cofunctionalized-NPs containing polyclonal antibodies and unreleased from the NPs and transferred to the detection region where thea scanometric detection protocol. (F) The barcode molecules attachUniversal NP probes are attached to the barcode DNA. (H) The univspectrum. The red-colored channels represent the target capture regionregion of the device. Dark blue lines are pneumatic control channels u

108 JALA April 2010

unique microfluid scaling and adjusts for the constraints ofLab-On-Chip (LOC) systems.7

Chip-based BCAs developed thus far have used MMPs.However, there are a number of issues associated with exe-cuting this approach on chip. For example, because of thelarge volume difference between MMPs and the NP probes,it is very easy for NPs to become trapped in the packedbed of MMPs. In practice, this unintentional trappingincreases the false-positive rate. Removing nonspecificallytrapped AuNPs is not practical as it requires extensive washprotocols and substantially increases assay time (by nearly30 min). Furthermore, the use of MMPs requires the use ofswitchable electromagnets for the on-chip instrument,increasing the complexity and cost of the system.

A simple alternative is to directly pattern the monoclonalantibodies on the walls of the microfluidic channels7 (Fig. 1).In the presence of targets, the NPs would be bound to thechannel wall through the target linker. Instead of formingan MMPetargeteNP-sandwich in solution and then pullingthe sandwich to the reactor sidewall using a magnetic field,the sandwich is formed at the reactor wall. This new SI-BCA

of the capture region are coated with antibodies. (B) Samples arethe antibodies on the channel walls. (D) The target proteins are

ique barcode DNA oligonucleotides. (E) The barcode DNA is thencomplementary sequence is patterned. Steps from F to H illustrate

to the complementary sequences in the appropriate regions. (G)ersal probes are silver stained to facilitate visualization in the visible, whereas the green-colored channels mark the barcode detectionsed for directing the flow of fluid.

Original Report

protocol eliminates the need to use MMPs and reduces false-positive readings in microfluidic devices. It also reduces thecomplexity of the microfluidic system by eliminating the needfor creating a magnetic field on demand.

Figure 2. Schematic diagram of the E-gap biodetection schemeimplemented in a microfluid channel.

Electronic-Gap-Based Assay

The BCA can be quantified using a number of methods, in-cluding optical scattering,15 electric gaps,3 andRaman scatter-ing. Electrical detection does not require bulky opticalcomponents and should result in less energy-hungry instru-ments. Using the dip pen nanolithography (DPN) technique,capture single-stranded DNAs (ssDNAs) are written inside5-� 10-mmelectronic gaps (E-gaps) on substrates (Fig. 2). TheDPN-functionalizedE-gaps can specifically hybridize to targetssDNAs in solution. Successful hybridization of the captureetarget DNA complex is detected by the use of AuNPs carry-ing ssDNA, which also hybridize to the target ssDNA, fol-lowed by silver enhancement. The drop of resistance acrossthe gaps because of the formation of metal nanoparti-cleeDNA complexes is measured over time and comparedagainst characteristics of control gaps, which are either left un-functionalized or functionalized with noncomplementary cap-ture ssDNA. This technique has potential for high-densitymultiplexed DNA assay chips. Multiplex detection of two dif-ferent target ssDNAs in solution using DPN-functionalizedelectrical gaps on the same chip is demonstrated. The lowestdetection limit is 10 pM. It should be noted that this numberis lower than what was reported by Park et al. in Ref.3 Thedifference is attributed to changes in the protocol (i.e., DPNfunctionalization vs droplet functionalization). We believethere is room for improvement in the future.

Pipetting is usually used to spot capture ssDNA moleculesinside the electrical gaps of substrates. Spotting by pipettingmay result in a large size functionalized area, typically100� 100 mm. Such large sizes of functionalization area arenot amenable for the development of high-density multi-plexed detection chips and consumes large amount of captureoligonucleotide material. Using pipette spotting, it is impos-sible to produce functionalization area below 10� 10 mm.Therefore, it is advantageous to develop new functionaliza-tion techniques that overcome the aforementioned draw-backs of spotting methodology.16

DPN is an emerging technique to fabricate nanoscalechemical patterns.17,18 It uses a sharp scanning probe, oran array of scanning probes, to transfer chemical ink ontosolid substrates. These inks include small organic molecules,peptides, proteins, oligonucleotides, and inorganic solegel.In this technique, the atomic force microscope (AFM) tipis brought into close proximity to the substrate under properconditions. Ink biomolecules transport from the tip of anAFM to a substrate surface via a water meniscus. The typicaldimension of biomolecule patterns on the substrates fabri-cated by DPN is micron or submicron size. Therefore,DPN can easily produce precise and user-defined patternsinside confined, micron-sized electrical gaps.

Figure 3 is a representative SEM image of an electrode gapwith complementary DNA hybridization [LT114 chipDNAeLT114 target DNAeAuNP probe] complexes after

JALA April 2010 109

Figure 4. Resistance change across a gap with respect for time.The gap with complementary functionalized DNA exhibits themost rapid change of resistance.

Original Report

15-min silver staining.A currentevoltage curve of the filled gapindicates that the resistance of the filled gap is 11.76 U. The re-sistance measurement of parallel control gaps containing non-complementary LT68 chipDNAor no chip DNAwas infinity,indicating that properDNAcomplexes did not form andnano-particle-based, silver-enhanced DNA detection did not occur.

We investigated the effect of silver staining time on thedifferentiation of the detection gap and the control gaps next.The resistance of each gap and the differentiation among gapsis a function of staining time. We conducted a silver-stainingtime-course study formore than 30 min (Fig. 4). The chips usedin the study contained twogaps functionalizedwithLT114 chipDNA; one gap functionalized with LT68 chip DNA and onegap with no chip DNA functionalization. Solution containingLT114 target DNA (at a concentration of 10 nM) was used inthis time-course study. It can be observed from Figure 4 thatwhen a proper nanoparticle-containing DNA hybridizationcomplex is formed ([LT114 chip DNAeLT114 targetDNAeAuNP probe]), a measurable drop in resistance is seenfrom 6 to 12 min silver-staining time.During this time, the con-trol gaps containing noncomplementary chipDNA (LT68 chipDNA) or no DNA exhibit infinity resistance readings. In26 min, nonspecific silver-staining of the gap containing LT68chip DNA occurs as indicated by a drop in resistance, whereasthe gap with no chip DNA shows no silver staining or measur-able resistance even at 30-min staining time. This study showsthe optimal time for silver staining to differentiate detectiongaps and control gaps.Understaining (e.g., less than6 min)willnot differentiate various gaps, whereas overstaining (e.g., morethan 20 min) will result in nonspecific detection as well.

Mechanical Resonance Detection

The final step of the nanoparticles biodetection is a silverstaining, which increases the mass of the overall chemistryassembly by a significant amplification factor. We are there-fore interested in a mass-based detection method. One suchpossibility is to measure mass loading on a resonant

Figure 3. SEM micrograph of an E-gap after silver staining toclose the circuit gap.

110 JALA April 2010

cantilever and observe the frequency shift. Such a techniquecan be extremely useful compared with optical- or electro-magnetic wave based techniques. It may also provide greaterdynamic range when an array of cantilevers with differentsizes and resonant frequencies are used (Fig. 5). Comparedwith the E-gap-based technique, the frequency shift and massloading may yield more quantitative measurement.

An array of cantilevers has been realized, made of bothsilicon nitride and single crystal silicon (Fig. 5C, D). Initialtesting results using an array of single crystal silicon probeare shown in Figure 5EeG. Initial results show promise oflow detection limit (10 fM). More results will be discussedin the future.

MICROFLUID DEVELOPMENT

Advancement of microfabrication technology in the past fewdecades has enabled miniaturization and large-scale integra-tion of complex systems. This has resulted in the availabilityof such products as analog/digital integrated circuits andpowerful computers at affordable prices. The effects of min-iaturization and integration reach far beyond the semicon-ductor industry. Micro- and nanoscale technologies areincreasingly sought for purposes outside traditional elec-tronic applications.

One such example is the microfluidic LOC. Microfluidictechnology promises to automate macroscale, benchtop lab-oratory protocols and encapsulate them in low cost, portablesystems. These systems benefit from reduced consumption ofexpensive reagents, precise manipulation of small volumes offluid as low as a few picoliters, batch fabrication, and theability to analyze a sample closer to the length-scale of thesubjects of interest (e.g., cells, protein, DNA). Important ex-amples of LOC applications include sample preparation, cellmanipulation, biomolecular separation,19,20 the PCR,19,21

immunoassay-based detection,22,23 and hybridizationarrays.23,24 Hence, LOCs have the potential to dramaticallychange the way biochemical analysis is performed for clinical

Figure 5. A mechanical BCA assay. (A) Principle of mass loading and frequency shift. (B) Detailed view of molecular attachment to thecantilever. (C) Optical micrograph of a silicon nitride cantilever array. (D) Optical micrograph of a single crystal silicon cantilever array.(EeG) SEM of cantilever surfaces after exposure to 10 pM, 1 fM, and control fluid. (H) A typical frequency shift plot.

Original Report

diagnostics, environmental monitoring, pharmaceutical drugdiscovery, and chemical synthesis.

To successfully build an LOC, one must take into accounta variety of different concerns, including biochemicalcompatibility, channel surface passivation/functionalization,optical transparency, ease of microfabrication, system inte-gration, and the cost of development. Significant time andexpertise are required to resolve often nontrivial issues.

A gap currently exists between the developers and poten-tial users of microfluidic chips. Potential users, especiallythose in life sciences, generally do not have the means tomanufacture or purchase custom microfluidic systems.Custom systems incur substantial costs because of both

low-volume production and the long development time asso-ciated with creating highly functional devices.

From the developers’ perspective, two prevailing practicesin LOC development contribute to the difficulties of buildingsuch a system. First, existing systems often use a monolithicapproach, where chemical reactors, sensors, and actuatorsare integratedona single chip.This requires amicrofabricationprocess common to all components such that functionalitymay have to be compromised to build the device. Second, thecomponents typically reside on a single plane, creating a needfor elaborate channel routing to interconnect these compo-nents. Modifying one portion of monolithic and planar sys-tems frequently entails rebuilding the entire system.

JALA April 2010 111

Figure 6. (A) Schematic diagram of a representative FBB consist-ing of electromechanical components and through-wafer fluidicconnections. (B) A complete LOC can be built by bonding a passivefluidic chip with an FBB. The inset is a perspective view emphasiz-ing the fluidic communication between components on two levels.(C) Different functions may be realized by deploying differentpassive chips on the same FBB. (D) Multiple chips can be intercon-nected to form a larger system at the MCM level.

112 JALA April 2010

Original Report

Our approach to narrow the gap lies in the introduction ofa system-level microfluidic architecture that allows for rapidcustomization with low cost using materials satisfactory toboth users and developers. The system reaches these goalsby circumventing the obstacles encountered in conventionalapproaches.

We developed a new LOC architecture that is conceptual-ized on two levels: A single chip level and a multiple chipmodule (MCM) system level (Fig. 6). At the individualchip level, a multilayer approach segregates componentsbelonging to two fundamental categories: passive fluidiccomponents (channels and reaction chambers) and activeelectromechanical control structures (sensors and actuators).This distinction is explicitly made to simplify the develop-ment process and minimize cost. Components belongingto these two categories are built separately on differentphysical layers and can communicate fluidically via cross-layer interconnects (Fig. 7). The chip that hosts the electro-mechanical control structures is called the microfluidicbreadboard (FBB). A single LOC module is constructedby attaching a chip comprising a custom arrangement offluid-routing channels and reactors (passive chip) to theFBB. In one representative embodiment, two fluid layersmade of elastomers are interfaced through a silicon chipwith vertically etched holes. Tight seal is ensured throughnormal contact pressure and deformation of the elastomerlayer. Many different LOC functions can be achieved usingdifferent passive chips on an FBB with a standard resourceconfiguration. Multiple modules can be interconnected toform a larger LOC system (MCM level). Applications ofbiomolecule detection (DNA) and heavy metal ion detectionhave been demonstrated in chips made using this modulararchitecture.14

CONCLUSIONS

The AuNP is a versatile enabler of novel high-sensitivity andhigh-selectivity chemical assays for medical applications. Wehave developed various assays that are custom optimized forthe microfluid system implementations. Some assays showunique performance characteristics that rival those of bench-top systems. Future work will focus on three performanceaspects: (1) sensitivity and selectivity should be 100e1000better than the best laboratory assay currently available inhospitals, (2) the assay must be reliable and repeatable, soit can be used for point of care and remote field applications,and (3) the assay, together with chips and control/readoutsystems, must be made at low cost.

ACKNOWLEDGMENTS

Work discussed in this review has been supported by the United States

National Science Foundation (Nanoscale Science and Engineering Center),

The Department of Defense (DARPA Symbiotic Project), and the National

Institutes of Health (NIH Center for Excellence in Cancer Nanotechnology).

Competing Interests Statement: The authors certify that they have no relevant

financial interests in this manuscript.

Figure 7. First-generation FBB implementation. (A) The systemwas assembled by reversibly bonding a passive polydimethylsilox-ane (PDMS) chip to the FBB, which consisted of an active PDMSchip bonded to an oxidized silicon wafer with through-wafer holes.(B) Pneumatically actuated valves were formed at the crossing ofthe pneumatic control (red) and fluid (blue) channels on the activechip. Single valves may be used to control sample loading,whereas multiple valves can be used for multipath switching. (C)An optical micrograph of a single LOC. Holes were punched inthe PDMS chip to provide pneumatic connection ports to thevalve control channels. (D1) Cross-sectional view of the LOCemphasizing the operation of a single valve. An empty channel is(D2) cutoff by a thin PDMS membrane actuated via pneumaticpressure. (D3) Fluid may be filled up to the closed valve as airescapes through the PDMS. (D4) Releasing the pressure in thepneumatic control line opens the valve and allows the fluid tocontinue flowing.

Original Report

REFERENCES

1. Nam, J.-M.; Stoeva, S. I.;Mirkin,C.A.Bio-bar-code-basedDNAdetection

with PCR-like sensitivity. J. Am. Chem. Soc. 2004, 126, 5932e5933.

2. Whitesides, G. M. Nanoscience, nanotechnology, and chemistry. Small

2004, 1(2), 172e179.

3. Park, S.-J.; Taton, T. A.;Mirkin, C. A. Array-based electrical detection of

DNA with nanoparticle probes. Science 2002, 295(5559), 1503e1506.

4. Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Scanometric DNA array de-

tection with nanoparticle probes. Science 2000, 289(5485), 1757e1760.

5. Georganopoulou, D. G.; et al. Nanoparticle-based detection in cerebral

spinal fluid of a soluble pathogenic biomarker for Alzheimer’s disease.

Proc. Natl. Acad. Sci. U.S.A. 2005, 102(7), 2273e2276.

6. Goluch, E. D.; et al. A bio-barcode assay for on-chip attomolar-sensitiv-

ity protein detection. Lab chip 2006, 6, 1293e1299.

7. Goluch,E.D.; et al.Amicrofluidic detection systembasedupona surface im-

mobilized biobarcode assay. Biosens. Bioelectron. 2009, 24(8), 2397e2403.

8. Li, S.; et al. Dip pen nanolithography functionalized electrical gaps for

multiplexed DNA detection. Anal. Chem. 2008, 80(15), 5899e5904.

9. Khoo, M.; Liu, C. Micro magnetic silicone elastomer membrane actua-

tor. Sens. Actuators A Phys. 2001, 89(3), 259e266.

10. Liu, C. Foundations of MEMS. Illinois ECE Series. Upper Saddle River,

NJ: Prentice-Hall; 2005.

11. Ryu, K.; Liu, C. Micro magnetic stir-bar mixer integrated with parylene

microfluidic channels. Lab chip 2004, 4(6), 608e613.

12. Ryu, K. S.; et al. A two-terminal longitudinal hotwire sensor for moni-

toring the position and speed of advancing liquid fronts in microfluidic

channels. Appl. Phys. Lett. 2006, 88, 104104.

13. Ryu,K.; et al. Amethod for precision patterning of silicone elastomer and its

applications. IEEE/ASMEJ.Microelectromech.Syst.2004,13(4), 568e575.

14. Shaikh,K.A.; et al. Amodularmicrofluidic architecture for integratedbio-

chemical analysis.Proc.Natl. Acad. Sci.U.S.A. 2005,102(28), 9745e9750.

15. Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Nanoparticle-based bio-bar

codes for the ultrasensitive detection of proteins. Science 2003,

301(5641), 1884e1886.

16. Demers, L. M.; et al. Direct patterning of modified oligonucleotides on

metals and insulators by dip-pen nanolithography. Science 2002,

296(5574), 1836e1838.

17. Hong, S.; Mirkin, C. A. A nanoplotter with both parallel and serial

writing capabilities. Science 2000, 288(5472), 1808e1811.

18. Hong, S.; Zhu, J.; Mirkin, C. A. Multiple ink nanolithography: toward

a multiple-pen nano-plotter. Science 1999, 286(5439), 523e525.

19. Lagally, E. T.; Medintz, I.; Mathies, R. A. Single-molecule DNA ampli-

fication and analysis in an integrated microfluidic device. Anal. Chem.

2001, 73(3), 565e570.

20. Han, J.; Craighead, H. G. Separation of long DNAmolecules in a micro-

fabricated entropic trap array. Science 2000, 288(5468), 1026e1029.

21. Kopp, M. U.; de Mello, A. J.; Manz, A. Chemical amplification: contin-

uous-flow PCR on a chip. Science 1998, 280(5366), 1046e1048.

22. Wang, J.; Ibanez, A.; Chatrathi, M. P. On-chip integration of enzyme

and immunoassays: simultaneous measurements of insulin and glucose.

J. Am. Chem. Soc. 2003, 125(28), 8444e8445.

23. Selvaganapathy, P. R.; Carlen, E. T.; Mastrangelo, C. H. Recent prog-

ress in microfluidic devices for nucleic acid and antibody assays. Proc.

IEEE 2003, 91(6), 954e975.

24. Tillib, S. V.; Mirzabekov, A. D. Advances in the analysis of DNA

sequence variations using oligonucleotide microchip technology. Curr.

Opin. Biotechnol. 2001, 12(1), 53e58.

JALA April 2010 113